PAX transcription factors usually play an indispensable role in various developmental processes. For instance PAX5 plays an important role in B cell development and PAX8 has a key role in the differentiation of thyroid epithelium . However, we have previously shown that PAX5 and PAX8 are differentially expressed in lung cancers; while PAX5 expression is restricted to SCLC cells, PAX8 expression is apparent more in NSCLC cell lines . Such out-of-context expression underscores the loss of any normality in lung cancer. In this paper, we have shown that PAX8 is functional in NSCLC and is overexpressed in a majority of NSCLCs, irrespective of their sub-type, including their lymph node metastatic forms. As with PAX5, there appears to be strong physical as well as a functional link between PAX8 and the receptor tyrosine kinases MET and RON. A strong cellular colocalization between PAX8 and MET was observed and the loss of PAX8 expression in NSCLC cells also revealed a concomitant loss in the expression of MET and RON receptor kinases but not EGFR. With respect to functionality, PAX8 is very likely to significantly contribute to both lung tumor growth and metastasis as surmised from its role in cell viability and motility. Knockdown of PAX8 resulted in a significant but modest loss in cell viability, and further treatment with the MET inhibitor SU11274 had only a marginal additive effect.
Under physiological conditions, PAX8 is required for the normal development of the kidneys, thyroid gland, and the Mullerian system [13, 18, 19]. Recently, a systematic study of more than 1100 (normal and tumor) cases revealed that it was overexpressed in majority of the renal, ovarian, and endometrial cancers; whereas only one out of 100 lung cases turned out to be positive . This is clearly in contrast to the findings reported here. Our IHC analysis of archival lung tumor tissues unmistakably showed that the majority of large cell carcinoma (94%), adeno carcinoma (80%), squamous cell carcinoma (100%), and metastatic tumors in lymph nodes (93%) were positive for PAX8 expression (n = 254). Also, overexpression of PAX8 in endometrial cancer is associated with a poor prognosis [19, 21]. Although site directed loss-of-function mutants have been reported in hypothyroidism, no gain-of-function mutants have been reported for PAX8 in any cancers. Using genomic DNA extracts prepared from NSCLC archival tumor tissues, we failed to see any mutations in PAX8 genomic DNA (exons), while MET mutations were apparent (data not shown). In 36% of follicular thyroid cancers, there is a chromosomal translocation (2;3) (q13;p25) resulting in the formation of PAX8-PPAR-γ (peroxisome proliferator activated receptor-γ) fusion protein . While, in vitro experiments have clearly demonstrated the oncogenecity of the above fusion protein, recent studies in follicular thyroid carcinoma suggest a better prognosis in cases where the fusion protein was found to be overexpressed .
The silencing of PAX8 in NSCLC cells not only resulted in decreased levels of MET, but also that of RON. MET and RON are family members of the receptor tyrosine kinase and are important in the pathogenesis of a number of malignancies. The synergistic role of MET/RON is just beginning to be defined and their inhibition appears to be more important than inhibition of either one of the receptors. The findings here show that MET/RON could potentially be down regulated by decreasing expression by the PAX8. A recent PAX8 silencing study using rat FLT-6 thyroid cells revealed a total of 2815 genes modulated. A key finding of this study was that PAX8 is the primary regulator of thyroid morphogenesis and differentiation. In addition to genes that play a direct role in thyroid development and function, the authors identified several genes regulated by PAX8 that belonged to cell proliferation, apoptosis, tyrosine kinases, DNA replication, and anion transport. One of the genes positively regulated by PAX8 was MET. However the decrease in MET transcripts in PAX8 knockdown cells, although significant, was only 1.4 fold . In the present study, we observed a dramatic decrease in the protein expression levels of MET and RON. We previously showed the presence of a functional PAX binding site in MET (and also RON) proximal promoter region . It thus appears that PAX8 regulates the transcription of MET and RON but not EGFR. Similar to our earlier report with respect to PAX5 , we also observed a strong colocalization of PAX8 with active forms of MET and RON kinases, especially after the NSCLC cells were stimulated with HGF. The fact that the colocalization between PAX8 and the RTKs is conserved even in the simple nematode suggests a strong functional link. One possibility that is being currently tested is whether the active forms of MET and RON as feedback inhibitors of PAX8 transcription.
We have shown here clearly that PAX8 promotes cell motility and wound healing, a harbinger of its role in metastasis. Most likely, the above effect is through its ability to promote the transcription of both MET and RON RTKs. We and others showed that both MET and RON provide essential growth signals for lung tumor development and also play a key role in metastasis . It is not surprising that PAX8 knockdown results in significant loss in A549 cell viability; as such a role was demonstrated for PAX transcription factors including PAX8 in the promotion of cancer cell growth [12, 24, 25]. In order to rule out the vagaries of using one transformed cell line, A549, we also repeated the functional assays using H1993, another NSCLC cell line and investigated the effects of PAX 8 knockdown on loss of cell motility and viability. We have consistently observed that they were similar to that shown for A549 cells. For instance, knockdown of PAX8 in H1993 cells resulted in a loss in the ability to close the gap created in scratch assay by about 20% at 12 h that was comparable to that seen in PAX8 knockdown A549 cells in relation to respective scrambled RNA treated controls, although actual values were somewhat different. In terms of cell viability, PAX8 knockdown had a marginal inhibitory effect in both A549 and H1993 cells. Inhibition of MET using specific inhibitors is also known to adversely affect NSCLC cell viability and is the basis for several ongoing clinical trials aimed at testing the efficacy of MET inhibitors in NSCLC and other cancers . Our results using SU11274, an ATP-competitive small molecule inhibitor of the catalytic activity of MET in A549 cells, clearly support the above. However, a combinatorial approach of using SU11274 in PAX8 knockdown background failed to induce synergetic loss in A549 cell viability. Also in H1993 cells, it was the MET inhibitor (SU11274) that had maximum inhibitory effect on cell viability that was comparable to that observed in A549 cells. There was however no discernible synergy on loss of cell viability when the SU11274 was used in PAX8 knockdown H1993 cells. These observations substantiate our contention that PAX8 knockdown effects in NSCLC cells are consistent and due to the use of any select NSCLC cell line. The lack of synergy can be logically explained by the fact that a loss in PAX8 transcription factor activity results in a dramatic loss in MET expression, thereby making the use of SU11274 redundant, at least with respect to MET. Synergy in killing NSCLC is more likely to be achieved by combining PAX8 knockdown with a non-overlapping disparate approach such as cisplatin that forms DNA adducts .